Exosome purification device

Abstract

The utility model belongs to the field of biomedical engineering, and particularly relates to an exosome purification device. The device comprises an electric field mechanism and a purification mechanism, the electric field mechanism comprises a first electrode plate, a second electrode plate and a power supply, the purification mechanism is positioned between the first electrode plate and the second electrode plate, and the power supply is electrically connected with the first electrode plate and the second electrode plate, so that one of the first electrode plate and the second electrode plate is positively charged, and the other is negatively charged; the purification mechanism is provided with a purification tank, a collection structure, a lead-in structure and a lead-out structure, the collection structure is used for collecting purified exosomes, the lead-in structure is used for leading in an exosome reagent, and the lead-out structure is used for leading out the exosome reagent. According to the utility model, the exosomes are adsorbed to the bottom of the purification tank by utilizing an electric field, so that the recovery rate is increased, the exosome separation efficiency and the purity of the obtained exosomes are improved, and large-scale high-concentration exosome purification is realized.

Description

Technical Field

[0001] The utility model belongs to the technical field of biomedical engineering, and in particular relates to an exosome purification device. Background Art

[0002] Exosomes are small membrane vesicles released from cells. Enclosed by cell membranes, they contain cell-derived components such as proteins, nucleic acids, and lipids. They can be found in a variety of biological fluids, including plasma, urine, and saliva. Exosomes play a key role in intercellular communication, cargo transport, immune responses, and the pathogenesis of various diseases. Therefore, the efficient purification and isolation of exosomes is crucial.

[0003] Traditional methods for exosome purification include chromatography, centrifugation, and magnetic bead methods. Chromatographic separation of exosomes is susceptible to exosome loss due to varying particle sizes from different sources. Centrifugation can easily damage exosome integrity during repeated high-speed centrifugation to increase purity. While magnetic bead separation offers high purity, it is only suitable for small-scale laboratory use and cannot meet the large-scale clinical requirements. Purifying exosomes on a large scale remains a major challenge. Furthermore, traditional exosome purification methods rely on specialized laboratory equipment, which is not universally applicable and difficult to scale up. Furthermore, there are no readily available, user-friendly exosome purification devices on the market.

[0004] Exosomes possess several physical properties that enable them to migrate in an external electric field, enabling their efficient isolation. First, their nanoscale size makes their behavior in an electric field distinct from that of larger biomolecules, making their purification possible. Second, exosomes possess a negative surface charge, particularly due to the presence of phosphatidylserine (PS) residues, which causes them to migrate toward the positive electrode in an electric field, thus achieving directional movement. Furthermore, exosomes possess specific dielectric properties that induce dielectrophoretic forces, driving them toward the electrode and further accelerating their purification. By precisely controlling the intensity of the electric field, the migration efficiency of exosomes can be modulated, enabling precise control of the purification process. This electric field purification technique eliminates the need for chemical labeling or the use of specific antibodies, preserving the exosomes' native state and biological activity. Furthermore, this technique is applicable to exosomes from a variety of sources, including cell culture supernatants, blood, and other bodily fluids, offering a broad range of applications for exosome research. Finally, electric field purification technology can be combined with automated systems to achieve high-throughput separation, providing convenience for clinical applications and large-scale production, and is of great significance to the research and application of exosomes. Based on electric field purification technology, the utility model provides an exosome purification device. Utility Model Content

[0005] The purpose of the present invention is to solve the problems of the prior art and to provide an exosome purification device.

[0006] In order to achieve the above-mentioned purpose, the technical solution provided by the present invention is:

[0007] An exosome purification device includes an electric field mechanism and a purification mechanism, wherein the electric field mechanism includes a first electrode plate, a second electrode plate, and a power supply, the purification mechanism is located between the first electrode plate and the second electrode plate, and the power supply is electrically connected to the first electrode plate and the second electrode plate, so that one of the first electrode plate and the second electrode plate is positively charged and the other is negatively charged;

[0008] The purification mechanism includes an upper cover, a bottom plate, a collection structure, an introduction structure, and an export structure. A purification pool is provided on the bottom plate, and the size of the purification pool is smaller than the first electrode plate and the second electrode plate. The collection structure, the introduction structure, and the export structure are connected to the purification pool. The upper cover is installed on the side of the bottom plate where the purification pool is provided. The bottom plate is close to the positively charged electrode plate, and the upper cover is close to the negatively charged electrode plate. The collection structure is used to collect purified exosomes, the introduction structure is used to introduce the exosome reagent, and the export structure is used to export the exosome reagent. The purification mechanism is non-conductive.

[0009] Furthermore, the collection structure includes a first pipe and a suction assembly, one end of the first pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the suction assembly, the introduction structure includes a second pipe and a first suction and injection assembly, one end of the second pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the first suction and injection assembly, the export structure includes a third pipe and a second suction and injection assembly, one end of the third pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the second suction and injection assembly.

[0010] Furthermore, the bottom of the purification pool is provided with a plurality of protrusions.

[0011] Furthermore, it also includes a supporting mechanism, which includes a first supporting plate, a second supporting plate and a supporting column, the first supporting plate and the second supporting plate are connected by the supporting column, the first electrode plate is installed on the first supporting plate, and the second electrode plate is installed on the second supporting plate, and the supporting mechanism is non-conductive.

[0012] Furthermore, the bottom plate and the upper cover are made of resin.

[0013] Furthermore, the first electrode plate and the second electrode plate are made of aluminum.

[0014] Furthermore, the distance between the first electrode plate and the second electrode plate is less than or equal to 2 cm.

[0015] Furthermore, the voltage output by the power supply is 5-20V.

[0016] Compared with existing technologies, this utility model has the following significant advantages: 1) Utilizing an electric field to adsorb exosomes to the bottom of the purification pool improves the efficiency of exosome separation and the purity of the exosomes obtained, enabling the purification of large quantities of high-concentration exosomes. 2) Exosome reagents are introduced through an inlet and outlet structure, and multiple cycles are performed to ensure that each exosome is fully exposed to the electric field, thereby achieving efficient separation, improving the purity of the exosomes, and increasing the recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the overall design of an exosome purification device provided in the present utility model;

[0018] Figure 2 This is a schematic structural diagram of an exosome purification device in the present invention;

[0019] Figure 3 This is a schematic diagram of the bottom plate of the purification mechanism of an exosome purification device in the present invention;

[0020] Figure 4 This is a schematic diagram of the upper cover of the purification mechanism of an exosome purification device in the present invention;

[0021] Figure 5 The results of the particle size peak diagram before and after exosome membrane rupture in the embodiment of the present invention are shown;

[0022] Figure 6 Schematic diagram of the relationship between purification efficiency and voltage in the experimental results of an embodiment of the present utility model.

[0023] Explanation of the accompanying drawings: 1. First electrode plate, 2. Second electrode plate, 3. First support plate, 4. Second support plate, 5. Purification mechanism support plate, 6. Support column, 7. Purification mechanism, 8. Bottom plate, 9. Upper cover, 10. Collection tank, 11. Inlet tank, 12. Outlet tank, 13. First circular hole, 14. Second circular hole, 15. Third circular hole, 16. First pipeline, 17. Third pipeline, 18. Purification tank, 19. Strip protrusion. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The utility model provides an exosome purification device, such as Figure 1~Figure 2As shown, the device includes an electric field mechanism and a purification mechanism 7. The electric field mechanism includes a first electrode plate 1, a second electrode plate 2, and a power source. The purification mechanism 7 is located between the first electrode plate 1 and the second electrode plate 2. The power source is electrically connected to the first electrode plate 1 and the second electrode plate 2, so that one of the first electrode plate 1 and the second electrode plate 2 is positively charged and the other is negatively charged. In this embodiment, the first electrode plate 1 is negatively charged and the second electrode plate 2 is positively charged. In other embodiments, the first electrode plate 1 can be positively charged and the second electrode plate 2 can be negatively charged as needed.

[0026] In this embodiment, the first electrode plate 1 is placed on the upper side of the purification mechanism 7, and the second electrode plate 2 is placed on the lower side of the purification mechanism 7. In other embodiments, a support mechanism may be introduced to position the first electrode plate 1, the second electrode plate 2, and the purification mechanism 7, thereby increasing the distance between the first electrode plate 1, the second electrode plate 2, and the purification mechanism 7.

[0027] There are several key factors to consider for both the power supply and the electric field it creates:

[0028] Charge properties: The surface charge of exosomes affects their migration rate in an electric field. Ensure that the charge properties of the exosomes match the exosome purification conditions.

[0029] Equipment parameters: The parameter settings of the exosome purification device, such as electric field strength, pulse frequency, and processing time, need to be optimized according to the specific characteristics of exosomes.

[0030] Stability: The stability of exosomes in the electric field is also a consideration to ensure that the structure and function of exosomes are not destroyed during the purification process.

[0031] Safety: When operating the exosome purification device, safety procedures need to be followed to protect the safety of researchers and samples.

[0032] Any power supply that meets the above key factors can be adapted. This embodiment uses a DP305B DC regulated power supply.

[0033] In this embodiment, the support mechanism includes a first support plate 3, a second support plate 4, a purification mechanism support plate 5 and a support column 6. The first support plate 3, the second support plate 4 and the purification mechanism support plate 5 are connected by the support column 6. The first electrode plate 1 is installed on the lower side of the first support plate 3. The second electrode plate 2 can be installed on the upper side of the second support plate 4 or on the lower side of the purification mechanism support plate 5. The purification mechanism 7 is installed on the purification mechanism support plate 5. The support mechanism is non-conductive.

[0034] It should be noted that the distance between the first electrode plate 1 and the second electrode plate 2 is less than or equal to 2 cm, and the distance depends on the voltage output by the power supply. Depending on the required distance between the first electrode plate 1 and the second electrode plate 2, it is optional to set a support mechanism.

[0035] The following factors should be considered when selecting materials for the electric field mechanism:

[0036] Stability: The electric field system should be stable and reliable to ensure repeatability and consistency.

[0037] Temperature control: In some cases, it may be necessary to control the temperature during the purification process to prevent thermal effects on the exosomes.

[0038] Adjustable: The direction of the electric field can be adjusted according to the experimental design to achieve different separation effects.

[0039] Ease of operation: Electric field equipment should be easy to operate and convenient for researchers to use.

[0040] Safety: The electric field mechanism should comply with safety standards to protect the safety of operators and samples.

[0041] Compatibility: The electric field mechanism should be compatible with existing laboratory equipment and workflows.

[0042] Cleaning and disinfection: The electric field mechanism should be easy to clean and disinfect to prevent cross contamination.

[0043] In this embodiment, the first electrode plate 1 and the second electrode plate 2 are aluminum sheets.

[0044] The purification mechanism 7 includes an upper cover 9, a bottom plate 8, a collecting structure, an import structure and an export structure. The collecting structure includes a first pipe 16 and a suction assembly. The import structure includes a second pipe (not shown in the figure) and a first suction and injection assembly. The export structure includes a third pipe 17 and a second suction and injection assembly. The suction assembly, the first suction and injection assembly and the second suction and injection assembly can adopt syringes, suction pumps and other equipment with suction and injection functions. It should be noted that the first suction and injection assembly and the second suction and injection assembly can be set separately or the same. When they are the same, a suction pump can be used to circulate the reagent from the export port to the import port. When set separately, the reagent is extracted from the export port and pressed into the import port. It can be extracted and then sent to the pressing side from the outside, or it can be circulated by mutual squeezing of the syringes. At this time, the import structure and the export structure are not a single import or export, but a circulating inlet and outlet. As Figure 3As shown, the bottom plate 8 is provided with a purification pool 18, a collection tank 10, an inlet tank 11 and an outlet tank 12, and the collection tank 10, the inlet tank 11 and the outlet tank 12 are connected to the purification pool 18. The size of the purification pool 18 is smaller than the first electrode plate 1 and the second electrode plate 2, so that the purification pool 18 is completely placed in the electric field. Figure 4 As shown, the upper cover 9 is provided with a first circular hole 13, a second circular hole 14 and a third circular hole 15. The upper cover 9 is mounted on the upper side of the base plate 8. The position of the collecting tank 10 corresponds to the position of the first circular hole 13, the position of the introducing tank 11 corresponds to the position of the second circular hole 14, and the position of the exporting tank 12 corresponds to the position of the third circular hole 15. The first pipe 16 is inserted into the first circular hole 13 and connected to the collecting tank 10 for collecting purified exosomes. The second pipe is inserted into the second circular hole 14 and connected to the introducing tank 11 for introducing the exosome reagent. The third pipe 17 is inserted into the third circular hole 15 and connected to the exporting tank 12 for exporting the exosome reagent.

[0045] During the purification process, the exosome reagent circulates inside the purification pool 18. The exosome reagent is introduced through the second pipe and discharged through the third pipe 17 for multiple cycles to ensure that each exosome is fully exposed to the electric field, thereby achieving efficient separation, improving the purity of the exosomes, and increasing the recovery rate.

[0046] The collecting tank 10 , the inlet tank 11 and the outlet tank 12 may be located at any position of the bottom plate 8 , wherein the inlet tank 11 and the outlet tank 12 are optimally located near two diagonal corners of the purification tank 18 .

[0047] The bottom of the purification pool 18 is also provided with a number of protrusions to reduce the flow rate. The protrusions can be of any shape, such as strip-shaped protrusions 19. The ends of the strip-shaped protrusions 19 do not contact the inner wall of the purification pool 18. The provision of the strip-shaped protrusions 19 reduces the flow rate of the exosome reagent in the purification pool 18, allowing the exosomes to be adsorbed to the bottom of the purification pool 18 under the full action of the electric field, thereby improving the purification efficiency.

[0048] The following points should be noted when selecting materials for purification mechanism 7:

[0049] Biocompatibility: The material needs to have good biocompatibility to avoid affecting the biological activity and structure of exosomes.

[0050] Non-adsorbability: The material should have low nonspecific adsorption to reduce the possibility of nonspecific binding of exosomes with other biomolecules, and also reduce the possibility of exosomes adsorbing to the bottom of the reaction pool and being unable to be stripped off.

[0051] Conductivity: The material should have appropriate conductivity to ensure uniform distribution of the electric field, allowing exosomes to migrate effectively and ensuring the purification effect.

[0052] Chemical stability: The material needs to be chemically stable enough to resist the chemical reagents used in the experiment, including buffer solutions, saline solutions, etc.

[0053] High temperature resistance: In case the purification process requires heating, the material should be able to withstand a certain temperature range without deformation or releasing harmful substances.

[0054] Transparency: In order to timely observe the status of the exosome purification reaction pool and check whether the device is blocked or leaking, the material needs to have good transparency so that problems can be discovered and remedied in time.

[0055] Mechanical strength: Equipment may encounter bumps during storage, transportation, assembly and experimentation. To avoid accidents, the material should have sufficient mechanical strength.

[0056] Corrosion resistance: The material should be resistant to common chemical corrosion, including acids, bases and organic solvents.

[0057] Easy to clean and disinfect: Materials should be easy to clean and disinfect to prevent cross contamination.

[0058] Anti-pollution: The material should not be prone to the growth of microorganisms to ensure the sterility of the purification environment and protect the exosomes from damage.

[0059] Cost: Material purchases should be controlled within an appropriate cost, and in case of unexpected situations, additional purchases can be made in a timely manner.

[0060] Compatibility: The material should be compatible with other components in the electric field purification system.

[0061] Safety: Materials should meet safety standards and be non-toxic and non-radioactive.

[0062] Machinability: The material should be easy to process into the required shape and size to meet the experimental requirements.

[0063] In this embodiment, the bottom plate 8 and the upper cover 9 of the purification mechanism 7 are non-conductive and are made of resin material.

[0064] The power supply output voltage is 5-20V. By adjusting the voltage of the DC regulated power supply, the electric field strength can be precisely controlled, thereby optimizing the migration efficiency and purification of exosomes. Experiments may require testing different voltage conditions to determine the optimal purification parameters. After purification, exosome samples are collected from the grooves and subjected to subsequent characterization analyses, such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot (WB), to verify the purity and bioactivity of the exosomes. The following experiments demonstrate a relationship between purification efficiency and voltage settings.

[0065] The experimental steps are detailed as follows:

[0066] (1) Prepare exosome samples: Collect and prepare exosome samples to be purified.

[0067] (2) Adjust the equipment: Since exosomes are negatively charged, a DC regulated power supply is used to provide a unidirectional stable electric field. Connect the positive pole of the power supply to the bottom of the ionizer and the negative pole to the top of the inner wall of the ionizer.

[0068] (3) Set experimental parameters: Set a series of different voltage and current conditions for subsequent experiments. According to the experimental group, adjust the voltage parameters to 5V, 10V, 15V, and 20V.

[0069] (4) Perform electric field purification: Perform electric field purification of exosomes under each set voltage and current condition. During this process, the exosome reagent should be pushed into the syringe at a constant speed as much as possible, and the exosome reagent flowing through the electric field should be collected during this period, and the flow should be repeated through the purification reaction pool several times.

[0070] (5) Collect and label exosome samples under different conditions: Ensure that the samples under each condition are accurately labeled and recorded. And classify and label the voltage parameters and experimental time during the experiment.

[0071] (6) Characterize and analyze each sample: Contact a professional testing agency to use TEM, NTA, WB and other technologies to conduct multi-dimensional monitoring and evaluation of exosome samples.

[0072] (7) Purity assessment: Specific exosome marker proteins were detected by Western blotting to assess sample purity. The concentration and recovery rate of exosomes were assessed by NTA or flow cytometry.

[0073] (8) Record data: Record experimental conditions, purification results, and any observed phenomena in detail. Analyze the data provided by the institution in real time. Compare the purity and recovery rates under different conditions and explore which conditions are more effective for purification.

[0074] (9) Optimize parameters: Based on the data analysis results, adjust the voltage and current parameters to optimize the purification effect.

[0075] (10) Repeat the experiment until the optimal conditions are found: Repeat the experiment using the optimized parameters until satisfactory purity and recovery are obtained.

[0076] (11) Complete the experiment: After determining the optimal voltage and current settings, end the experiment.

[0077] (12) Record the experimental report: Write a detailed experimental report including the experimental steps, results and conclusions.

[0078] The experimental steps may be modified in some details according to the actual situation. The time for testing the results of the exosome purification effect in this experiment will be adjusted accordingly based on objective conditions and professional institutions.

[0079] Exosome particle size analysis:

[0080] Remove an appropriate amount of exosomes and dilute them to the appropriate multiple. First, perform a performance test with a standard sample before loading the exosome sample. Be careful to perform a gradient dilution to prevent sample clogging of the injection needle. Once the sample is tested, the instrument will provide information on the particle size and concentration of the exosomes.

[0081] Table 1 shows the results of the exosome particle size analysis of the experimental samples. It can be seen from Table 1 that as the voltage increases, the average particle size of the exosomes decreases and the concentration increases.

[0082] Table 1 Exosome particle size analysis results

[0083] Sample name Average particle size (nm) Concentration (Particle / mL) 5V-Ninth Hospital 165.8 9.9E+8Particle / mL 10V-Ninth Hospital 151.2 1.6E+9 Particle / mL 15V-Ninth Hospital 148.4 4.5E+9 Particle / mL 20V-Ninth Hospital 137.4 2.2E+10Particle / mL

[0084] Exosome membrane rupture processing:

[0085] Take out the exosomes and dilute them to the appropriate multiple. Prepare 10% TritonX-100 and add 10% TritonX-100 to The above exosomes were mixed, incubated on ice for 1 hour, and then directly tested on the instrument.

[0086] Table 2 shows the concentration and purity of the exosomes before and after membrane rupture of the experimental samples. Figure 5~Figure 6 It can be seen that as the voltage increases, the purity of exosome extraction increases.

[0087] Table 2 Concentration of exosomes before and after membrane rupture

[0088] Sample name Concentration before membrane rupture (Particle / mL) Concentration after membrane rupture (Particle / mL) purity(%) 5V-Ninth Hospital 9.9E+8Particle / mL 2.1E+8 Particle / mL 78.8 10V-Ninth Hospital 1.6E+9 Particle / mL 1.8E+8Particle / mL 88.7 15V-Ninth Hospital 4.5E+9 Particle / mL 4.0E+8Particle / mL 91.1 20V-Ninth Hospital 2.2E+10Particle / mL 1.5E+9 Particle / mL 93.2

[0089] The above-described embodiments merely represent one or several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the utility model patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present utility model patent shall be determined by the appended claims.

Claims

1. An exosome purification device, characterized in that: The device comprises an electric field mechanism and a purification mechanism, wherein the electric field mechanism comprises a first electrode plate, a second electrode plate and a power supply, the purification mechanism is located between the first electrode plate and the second electrode plate, and the power supply is electrically connected to the first electrode plate and the second electrode plate, so that one of the first electrode plate and the second electrode plate is positively charged and the other is negatively charged; The purification mechanism includes an upper cover, a bottom plate, a collection structure, an introduction structure, and an export structure. A purification pool is provided on the bottom plate, and the size of the purification pool is smaller than the first electrode plate and the second electrode plate. The collection structure, the introduction structure, and the export structure are connected to the purification pool. The upper cover is installed on the side of the bottom plate where the purification pool is provided. The bottom plate is close to the positively charged electrode plate, and the upper cover is close to the negatively charged electrode plate. The collection structure is used to collect purified exosomes, the introduction structure is used to introduce the exosome reagent, and the export structure is used to export the exosome reagent. The purification mechanism is non-conductive.

2. The exosome purification device according to claim 1, characterized in that The collection structure includes a first pipe and a suction assembly, one end of the first pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the suction assembly. The introduction structure includes a second pipe and a first suction and injection assembly, one end of the second pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the first suction and injection assembly. The derivation structure includes a third pipe and a second suction and injection assembly, one end of the third pipe passes through the upper cover and is connected to the purification pool, and the other end is connected to the second suction and injection assembly.

3. The exosome purification device according to claim 1, characterized in that The bottom of the purification pool is provided with a plurality of protrusions.

4. The exosome purification device according to claim 1, characterized in that It also includes a support mechanism, which includes a first support plate, a second support plate and a support column. The first support plate and the second support plate are connected by the support column. The first electrode plate is installed on the first support plate, and the second electrode plate is installed on the second support plate. The support mechanism is non-conductive.

5. The exosome purification device according to claim 1, characterized in that The bottom plate and the upper cover are made of resin.

6. The exosome purification device according to claim 1, characterized in that The first electrode plate and the second electrode plate are made of aluminum.

7. The exosome purification device according to claim 1, characterized in that The distance between the first electrode plate and the second electrode plate is less than or equal to 2 cm.

8. The exosome purification device according to claim 1, characterized in that The voltage output by the power supply is 5-20V.

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